Pyrrole alkaloids from the fruiting bodies of edible mushroom Lentinula edodes

Nine pyrrole alkaloid derivatives, including four new ones (1–4), were isolated from the wild mushroom Lentinula edodes for the first time. Their chemical structures were determined using UV-Vis spectroscopy, IR spectroscopy, MS, NMR spectroscopy, and single-crystal X-ray diffraction techniques. Compound 1, a previously unreported bicylo-pyrrole aldehyde homologue, was found to be a major component, approximately 8.2 μg g −1 in the dry powder of L. edodes. Compound 1 showed cytotoxicity against SMMC-772 (IC50 15.8 μM) without any cytotoxic effect on LO2, a normal hepatic cell line; compounds 1 and 2 displayed weak immunosuppressive activities by inhibiting the proliferation of induced T cells; compound 3 showed inhibition activity on the proliferation of HaCaT cell line (IC50 25.4 μM) and weak antioxidant activity at a concentration of 50 μM.


Introduction
Edible mushrooms are a well-known healthy food for their unique nutritional properties, which have low calories but are rich in nutrients, such as vitamins and minerals. The mushroom Lentinula edodes, commonly known as shiitake, is cultivated for both its culinary and medicinal qualities, which has long been utilized as an edible mushroom in East Asia, and its uses were rst documented in an ancient Chinese book from the second century, titled Shen Nong Ben Cao Jing (The Divine Farmer's Materia Medica). Lentinan, a high molecular weight neutral polysaccharide puried from a hot water extract of L. edodes in 1969, 1 has been approved as an anti-tumor drug. Subsequently, a series of research was carried out to illustrate the mechanism of lentinan and more active polysaccharides and proteins were found. [2][3][4][5] However, unlike many progresses on biomacromolecule, only a few studies have been conducted to clarify the secondary metabolites (SMs) of the fruiting bodies of L. edodes, resulting in the isolation of some active compounds, such as eritadenine and polyacetylene molecules. 6 Therefore, to complete the possible utilization of L. edodes from the perspective of SMs, we studied SMs in this mushroom leading to the isolation of nine pyrrole alkaloids.
Alkaloids are a large group consisting of diverse subgroups of natural products that are most extensively studied in plants.
Pyrrole is of special interest in developing new bioactive drugs owing to their anticancer, antimicrobial, antiviral, antimalarial, antitubercular, anti-inammatory, and enzyme-inhibiting properties, 7-9 e.g., the biggest-selling drug of all time, atorvastatin, a HMG-CoA reductase inhibitor, is just a pyrrole. Indeed, before 2002, diverse pyrrole alkaloids were known mostly from vegetal or marine origin. In mushrooms, the discovery of sciodole from Tricholoma sciodes, 10 an alkaloid containing both pyrrole and indole moieties in its structure, led to the beginning of isolating pyrroles from mushrooms. Subsequently, a series of pyrroles were identied from different mushrooms, however, none of them were from L. edodes. 8,11 Herein, we report the isolation and structural elucidation of compounds 1-9 ( Fig. 1). Moreover, the potential activities of the isolated compounds 1-5 were evaluated, including inhibitory effects against ve human cancer cell lines, a keratinocyte cell

Results and discussion
Compound 1 was obtained as colourless crystals. The molecular formula C 10  , and a conjugated ketone (d C 201.5). Aer the structure assignment using the 1D NMR data, compound 1 was deduced to possess a bicyclic system for the remaining two degrees of unsaturation. All proton resonances were assigned to their respective carbons via the HSQC spectrum. The planner structure of compound 1 comprising two rings, A and B, was successfully established as follows (Fig. 2). The HMBC correlations from H-1 to C-2, from H 3 -10 to C-8/C-9, and from H 2 -9 to C-7 conrmed the presence of the B ring, which constructed a motif of 3-ethyl-pyrrole-1-carbaldehyde. In addition, A ring, a cyclopentenone, was also constructed using the HMBC correlations from H 2 -4 to C-2/C-3/C-5, and from H 2 -5 to C-3/C-6/ C-7. The fusion of rings A and B was identied at C-3 and C-7 based on the HMBC experiment (Fig. 2). Therefore, the structure of compound 1 was established as 3-ethyl-4-oxo-2,4,5,6tetrahydrocyclopenta[c]pyrrole-1-carbaldehyde, as shown in Fig. 1, which was further proved by X-ray single crystal experiments (Fig. 3). A comprehensive literature research showed that the architecture of 1 is simple but quite novel.

Conclusions
In summary, four new pyrrole alkaloid derivatives (1-4) and ve known ones (5-9) with various bioactivities were isolated from the wild edible mushroom L. edodes by alcohol (95%) extraction followed by ethyl acetate fractionation. Compound 1, bearing a novel cyclopenta[c]pyrrole-1-carbaldehyde scaffold, could inhibit the proliferation of SMMC-7721 without any cytotoxic effect on normal hepatocytes, showing the value for further research. Undoubtedly, this work not only enriches the ingredients of L. edodes, which is benecial for the public's knowledge of it, but also provides solid evidence for L. edodes used as a functional food.

Fungal material
Fruiting bodies of the wild mushroom L. edodes were purchased from Lijiang City, Yunnan Province, China in 2016, and were identied by the molecular evidence of ITS gene fragment (GenBank accession no. OQ680131), whose BLAST search result showed that the sequence was similar (>99%) to the sequence of L. edodes. A voucher specimen (no. HZYXG01) was deposited at the School of Pharmacy, Henan University of Chinese Medicine.
Single crystal X-ray diffraction data for 1: a block-like specimen of C 10 H 11 NO 2 , approximate dimensions 0.180 mm × 0.330 mm × 0.370 mm, was used for the X-ray crystallographic analysis on the Bruker D8 Quest instrument. The integration of the data using a monoclinic unit cell yielded a total of 14 437 reections to a maximum q angle of 74.55°(0.80 Å resolution), of which 1838 were independent (average redundancy 7.855, completeness = 98.9%, R int = 2.99%, R sig = 2.11%) and 1775 (96.57%) were greater than 2s(F 2 ). The nal cell constants of a = 6.7030(4) Å, b = 9.0452(6) Å, c = 15.0090(9) Å, b = 93.234(2)°, volume = 908.55(10) Å 3 , T = 273(2) K. Data were corrected for absorption effects using the multi-scan method (SADABS). The structure was solved and rened using the Bruker SHELXTL Soware Package, using the space group P121/n1, with Z = 4, m(CuKa) = 1.54178. The nal anisotropic full-matrix leastsquares renement on F 2 with 119 variables converged at R 1 = 3.97%, for the observed data and wR 2 = 11.07% for all data. The goodness-of-t was 1.056. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk (CCDC no. 2251050).   The ve human cancer cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Hyclone) and maintained at 37°C under 5% CO 2 in a humidied atmosphere. The keratinocyte cell line was HaCaT (FH0186) bought from Fufeng Biology (Shanghai, China), and this cell line was cultured in a DMEM medium containing 10% FBS. CCK-8 (Beijing Solarbio Science & Technology Co., Ltd, Beijing, China) was used to assess the cell viability. In brief, each well of a 96-well cell culture plate was seeded with 100 mL of adherent cells (1 ×10 5 cells per mL) and kept for 12 h for adherence and followed by the addition of the test compounds. However, 100 mL suspended cells (1 × 10 5 cells per mL) were seeded before being added the test compounds. Aer adding different concentrations of the test compounds, each cell line was incubated for 48 h. Cisplatin, Taxol, and methotrexate (MTX) were used as positive controls. Aer the incubation, each well was treated with CCK-8 (10 mL) and incubation was continued for 4 h at 37°C. Then, the 96-well cell culture plates were subjected to a measure of optical density at 450 nm with a 96-well microplate reader (SpectraMax iD3, Molecular Devices, LLC.). The IC 50 value for each compound was calculated by the Reed and Muench method. All experiments were performed in triplicate.
Immunosuppressive activities assay. The RPMI 1640 medium containing 10% FBS was used to culture the murine spleen cells. CCK-8 was used to assess cell viability. Compounds 1-5 were subjected to evaluate their inhibition on the proliferation of T and B lymphocytes. The fresh spleen cells were from BALB/c mice (7-9 weeks old, Beijing Huafukang Biotechnology Co., Ltd). The cell mixture was dispensed into 96-well plates (2 × 10 6 cells per mL), in the absence or presence of compounds (c = 20.0 mM), and were stimulated with ConA (5 mg mL −1 ) or LPS (15 mg mL −1 ) to induce T cell or B cell proliferative responses, respectively. These 96-well plates were maintained at 37°C under 5% CO 2 in a humidied atmosphere for 48 h in triplicate. Dexamethasone (DEX) (c = 10.0 mM) was used as a positive control. In the 96-well plates, the wells only lled with cells and ConA/LPS were assigned as the modal group (M), the wells just containing cells were described as the control group (Con), and the wells only containing culture medium were described as the blank group. 10 mL of CCK-8 was added to each well at the nal 4-5 h of culture, and then the absorbance (OD) values were measured with a microplate reader at 450 nm. Stimulation Index (SI) = (OD sample − OD blank )/(OD Con − OD blank ); inhibition rate = (1 − SI sample /SI M ) × %.
The DPPH free radical scavenging assay. This assay was modied slightly according to the previously reported method. 17 Compounds 1-5 (at a nal concentration of 50 mM) were mixed with DPPH (at a nal concentration of 100 mM) in ethanol. Aer incubation in the dark at 30°C for 1 h, the OD values were measured at 515 nm. Trolox (at a nal concentration of 25 mM) was used as the reference antioxidant. In the 96-well plates, the wells just containing DPPH were described as the control group (Con), and the wells only containing solvent were assigned as the blank group. The assay was carried out in triplicate. Antioxidant rate = [1 − (OD sample − OD blank )/(OD Con − OD blank )] × %.
Statistical analysis method. SPSS 22.0 soware was used for the statistical analysis of the data, which was expressed as X ± s. One-way ANOVA was used to compare the differences between groups. LSD or Dunnett's T3 test was used for pair comparison according to standard deviation values. P < 0.05 or P < 0.01 were considered statistically signicant.

Conflicts of interest
There are no conicts to declare.